Electron transfer reactions are crucial steps in a wide variety of biological transformations ranging from photosynthesis or aerobic respiration. Studies of electron transfer reactions in both chemical and biological systems have led to the development of a large body of knowledge and a strong theoretical base, which describes the rate of electron transfer in terms of a small number of parameters.
Electronic tunneling in proteins and other biological molecules occurs in reactions where the electronic interaction of the redox centers is relatively weak. Semiclassical theory reaction predicts that the reaction rate for electron transfer depends on the driving force (−ΔG°), a nuclear reorganization parameter (λ), and the electronic-coupling strength between the reactants and products at the transition state (HAB), according to the following equation:kET=(4π3/h2λkBT)1/2(HAB)2exp[(−ΔG°+λ)2/λkBT]
The nuclear reorganization energy, λ, in the equation above is defined as the energy of the reactants at the equilibrium nuclear configuration of the products. For electron transfer reactions in polar solvents, the dominant contribution to λ arises from the reorientation of solvent molecules in response to the change in charge distribution of the reactants. The second component of λ comes from the changes in bond lengths and angles due to changes in the oxidation state of the donors and acceptors.
Previous work describes using change in reorganization energy, λ, as the basis of novel sensors. See for example, U.S. Pat. Nos. 6,013,459, 6,013,170, 6,248,229, and 7,267,939, all herein incorporated by reference in their entirety. The methods generally comprise binding an analyte to or near a redox active complex. The redox active complex comprises at least one electroactive molecule and a capture ligand which will bind the target analyte, and the complex is bound to an electrode. Upon analyte binding, the reorganization energy of the redox active molecule is altered, thus changing the E0, and allowing detection.
It is an object of the present invention to provide composition and methods for the detection of target analytes using alterations in the solvent reorganization energy, such as utilizing cyano ligands with the transition metals of the biosensor, corresponding to changes in the E0 of redox active molecules.
The electromotive force (EMF) is the maximum potential difference between two electrodes of a galvanic or voltaic cell, where the standard hydrogen electrode is on the left-hand side for the following cell:
12Pt ElectrodeH2Aqueous Electrolyte10−3 M Fe(ClO4)3PtSolution10−3 M Fe(ClO4)2The EMF is called the electrode potential of the electrode placed on the right-hand side in the graphical scheme of the cell, but only when the liquid junction between the solutions can be neglected or calculated, or if it does not exist at all.
The electrode potential of the electrode on the right-hand side (often called the oxidation-reduction potential) is given by the Nernst equationEFe3+/Fe2+=EFe3+/Fe2+0+(RT/F)ln(aFe3+/aFe2+)This relationship follows from equation (2.21) when (μFe3−0+μFe2+0/F is set equal to EFe3+/Fe2+0 and the pH and ln pH2 are equal to zero. In the subscript of the symbol for the electrode potential the symbols for the oxidized and reduced components of the oxidation-reduction system are indicated. With more complex reactions it is particularly recommended to write the whole reaction that takes place in the right-hand half of the cell after symbol E (the ‘half-cell’ reaction); thus, in the present caseEFe3+/Fe2+≡E(Fe3++e=Fe2+)
Quantity EFe3+/Fe2+0 is termed the standard electrode potential. It characterizes the oxidizing or reducing ability of the component of oxidation-reduction systems. With more positive standard electrode potentials, the oxidized form of the system is a stronger oxidant and the reduced form is a weaker reductant. Similarly, with a more negative standard potential, the reduced component of the oxidation-reduction system is a stronger reductant and the oxidized form a weaker oxidant.
The standard electrode E0, in its standard usage in the Nernst equation, equation (1-2) is described as:
  E  =            E      0        +                            2.3          ⁢          RT                          n          ⁢                                          ⁢          F                    ⁢      log      ⁢                                    C            0                    ⁡                      (                          0              ,              t                        )                                                C            R                    ⁡                      (                          0              ,              t                        )                              Where E0 is the standard potential for the redox reaction, R is the universal gas constant (8.314 JK−1mol−1), T is the Kelvin temperature, n is the number of electrons transferred in the reaction, and F is the Faraday constant (96,487 coulombs). On the negative side of E0, the oxidized form thus tends to be reduced, and the forward reaction (i.e., reduction) is more favorable. The current resulting from a change in oxidation state of the electroactive species is termed the faradaic.
Previous work describes using conversion of functional groups attached to a transitional metal complex resulting in quantifiable electrochemical signal at two unique potentials, Eo1 and Eo2. See for example, U.S. Patent Publication Nos: US 2011 0033869 and US 2012-0181186, all herein incorporated by reference in their entirety. The methods generally comprise binding an analyte within a sandwich of binding ligands, which may have a functional tag, on a solid support other than the electrode. After target binding, a peroxide generating moiety or an intermediary enzyme and substrate are added, which generates hydrogen peroxide. The redox active complex is bound to an electrode and comprises a peroxide sensitive moiety (PSM). The peroxide generated from the enzyme system reacts with the PSM, removing a self-immolative moiety (SIM) and converting functional groups attached to a transitional metal complex resulting in quantifiable electrochemical signal at two unique potentials, Eo1 and Eo2.
While the forementioned methods for detection of target analytes using alterations in the solvent reorganization energy corresponding to changes in the Eo of redox active molecules or by measuring quantifiable electrochemical signals at two unique potentials Eo1 and Eo2 are useful for their intended purposes, improved robust redox active complexes that provide greater signal amplification, particularly where low concentrations of target analytes are involved, are desired.